This invention relates to a method of and apparatus for autologous plasma delipidation in animals (which term shall include humans) using a continuous flow system.
Cardiovascular diseases are responsible for more than half of all deaths in Australia and the United States and are frequent in most other industrialized countries. At present, an Australian or American male has a one in five risk of developing clinical evidence of coronary heat disease (CHD) before his 65th birthday.
One such disease is atherosclerosis which is characterised by focal fatty thickening in the inner aspects of large vessels supplying blood to the heart, brain and other vital organs. These lesions obstruct the lumen of the vessel and result in ischaemia of the tissue supplied by the vessel. Prolonged or sudden ischaemia may result in a clinical heart attack or stroke from which the patient may or may not recover.
It is believed that atherosclerosis begins in early childhood evolving through various stages before becoming clinically apparent in middle and late adult life. The earliest grossly recognizable intimal lesion is the fatty streak composed of lipid material, connective tissue and other substances. It is present in the aorta of many children less than 3 years of age. Fatty streaks become evident in the coronary arteries during the second decade, being seen in nearly all cases of CHD after 20 years of age. The frequency of coronary fatty steaks parallels the development of CHD. The most striking biochemical abnormality in human atherosclerosis is the accumulation of massive amounts of cholesteryl esters in the core of the atheromatous plaque.
The relationship between dietary lipid, serum cholesterol and atherosclerosis has long been recognised. In many epidemiological studies it has been shown that a single measurement of serum cholesterol has proved to be a significant predictor of the occurrence of CHD.
There seems little doubt concerning the relationship between elevations of plasma cholesterol and the development of premature CHD in humans. The fraction of cholesterol carried in low-density lipoproteins (LDL) appears to be particularly atherogenic. Thus, elevations of LDL and LDL-cholesterol apparently predispose individuals to an accelerated form of atherosclerosis.
As an individual grows larger and older, there is a net accumulation of cholesterol in body tissues, including the arterial wall. The body can rid itself of substantial quantities of cholesterol only through the liver, where cholesterol can be excreted in the bile and in the faeces. A mechanism has not yet been firmly established to explain how cholesterol from peripheral tissues, including the arterial wall, is transported to to the liver for removal. It has been suggested that high-density lipoprotein (HDL) plays a role in removing cholesterol from tissues and promoting reverse cholesterol transport to the liver.
Diet is the basic element of all therapy for hyperlipidaemia (excessive amount of fat in plasma). The use of diet as a primary mode of therapy requires a major effort on the part of physicians, nutritionists, dietitians and other health professionals.
If dietary modification is unsuccessful, drug therapy is an alternative. Several drugs, used singly or in combination, are available. However, there is no direct evidence that any cholesterol-lowering drug can be safely administered over an extended period.
Finally, a combination of both drug and diet may be required to reduce the concentration of plasma lipids. Hypolipidaemic drugs are therefore used as a supplement to dietary control.
Many drugs are effective in reducing blood lipids, but none work in all types of hyperlipproteinemia and they all have undesirable side-effects. There is no conclusive evidence that hypolipidaemic drugs can cause regression of atherosclerosis.
In view of the above, new approaches have been sought to remove LDL from the plasma of homozygotes and of those heterozygotes for whom oral drugs are not effective.
Plasmapheresis (plasma exchange) therapy has been developed and involves replacement of the patient's plasma with donor plasma or more usually a plasma protein fraction. This treatment can result in complications due to the possible introduction of foreign proteins and transmission of infectious diseases. Further, plasma exchange removes all the plasma proteins as well as VLDL, LDL and HDL and angiographic data suggest that HDL cholesterol is inversely correlated with the severity of coronary arterial lesions, as well as with the likelihood that these will progress. In the light of this, a technique designed to use the patient's own plasma and to conserve HDL during LDL removal would be desirable.
LDL apheresis can be achieved by various means, for example, by the absorption of LDL onto heparin-agarose beads (affinity chromatography) or the use of immobilized LDL-antibodies. Other methods presently available for the removal of LDL involve cascade filtration, absorption to immobilized dextran sulphate and LDL precipitation at low pH in the presence of heparin. Each method specifically removes LDL but not HDL.
LDL apheresis has, however, disadvantages. Significant amounts of other plasma proteins are removed during apheresis and to obtain a sustained reduction in LDL-cholesterol, LDL apheresis must be performed frequently (up to once weekly).
Therefore, there is still a need, using techniques other than diet and/or drug therapy, to achieve a reduction of plasma cholesterol and in particular LDL-cholesterol in homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia patients.
In response to the above problems, the present inventor has developed a biphasic solvent system for extraction of biological solution which attains complete removal of cholesterol, triglyceride, phospholipid and non-esterified fatty acids from plasma without protein denaturation. In particular, the solvent system has the following advantages:
(i) delipidation is complete in a short period of time; PA0 (ii) the treatment is mild enough so as not to affect ionic constituents or cause irreversible denaturation of proteins, including enzymes; PA0 (iii) the proteins, including the apolipoproteins (defatting lipoproteins), remain soluble in an aqueous phase, while an organic phase contains the dissolved lipids; PA0 (iv) the apolipoproteins dissolve lipids; PA0 (v) recovery of the aqueous phase after extraction is simple and very efficient; and PA0 (vi) the method is flexible i.e. it is applicable to small and large volumes of biological solutions as required. PA0 (a) drawing blood from the animal; PA0 (b) separating the plasma from the red blood cells; PA0 (c) delipidating the plasma with a lipid solvent; PA0 (d) remixing the delipidated plasma with the red blood cells; and PA0 (e) re-introducing the delipidated blood into the animal. PA0 (i) mixing the plasma with the liquid solvent; PA0 (ii) allowing the mixture to separate into an organic solvent/lipid phase and an aqueous delipidated plasma phase; and PA0 (iii) drawing off the organic phase. PA0 (a) means to draw blood from an animal; PA0 (b) means to separate the plasma from the red blood cells; PA0 (c) means to delipidate the plasma using a lipid solvent; PA0 (d) means to separate the delipidated plasma from the solvent; PA0 (e) means to remix the delipidated plasma with the red blood cells; and PA0 (f) means to re-introduce the delipidated blood into the animal.
In vitro studies have shown that a hyperlipaemic plasma--which looks turbid before delipidation--clears after delipidation using the above solvent system because the lipids are extracted from the plasma into an organic phase. The amount of lipid extracted from plasma can be measured after evaporation of the organic phase.
While the system discussed above has been successful in vitro in the experimental scale, it has not been suitable for the clinical delipidation of animal plasma.